In the diagnosis, treatment and management of patients with diabetes, sensitive instruments are employed for detecting one or more measurable characteristics of a patient blood sample. In a clinical laboratory environment there is a need to ensure that such instruments are performing properly. The use of control compositions having known characteristics is an accepted way to assure proper instrument performance. Though it is possible to use human whole blood as a control, it is not desirable due to stability considerations as well as availability. For example, in this context there would be a need for a ready supply of fresh diabetic whole blood. Another accepted approach is to synthesize a control composition that simulates relevant detectable characteristics measured by an instrument. An approach to one such synthetic control composition is illustrated in U.S. Pat. No. 7,361,513 (Ryan). Though such an approach yields useful control compositions, there remains a need for other such compositions. For example, there has been a longstanding need for a control that does not require diabetic blood as a starting material, that employs Alc synthesis entirely within a cell membrane, or any combination thereof. There is also a need for achieving a control that has in-situ synthesized Alc contained within a membrane in an amount of a nature so that it is detectable as such by a number of different instruments which may employ differing detection strategies. Hemoglobin (Hb) is a respiratory molecule found in red blood cells. It is responsible for transporting oxygen from the lungs to body cells and for transporting carbon dioxide from body cells to the lungs. Hemoglobin may be modified by the free glucose present in human plasma to form glycated hemoglobin (GHB). Hemoglobin Alc (Hb Alc, also referred to as Alc), constituting approximately 80 percent of all glycated Hb, is generated by the spontaneous reaction of glucose with the N-terminal amino group of the Hb A beta chain. The Hb Alc and the total glycated Hb values have a high degree of correlation, and either value may be used, for example in the management of treating diabetes. Formation of Hb Alc is slow but irreversible, and the blood level depends on both the life span of the red blood cells (average 120 days) and the blood glucose concentration. Therefore, Hb Alc represents the time-averaged blood glucose values over the preceding 2 to 3 months, and is not subject to wide fluctuations observed in blood glucose values. With respect to diabetes management, studies have shown that quality of life improves with decreasing levels of Hb Alc, and measurements every 3 to 6 months are recommended.
The determination of total hemoglobin is indicative of the oxygen-carrying capacity of whole blood. The numerous methods and devices for the determination of hemoglobin include both direct analysis, i.e., analysis without prior modification of the hemoglobin, and indirect analysis. It is important to accurately determine the total hemoglobin in the Hb Alc assay, because Alc is often reported as a fraction of the total hemoglobin. Multiple Hb Alc assay methodologies have been developed since late 1970s. One of the standard methods for measuring Hb Alc uses ionic-exchange high performance liquid chromatography (HPLC), which separates and analyzes Hb Alc and other minor Hb components from unmodified hemoglobin (Hb A0) based upon their differences in chemical charges. A second methodology for detection of Hb Alc is designed by immunoinhibition turbidimetric techniques. The Hb Alc assay in immunoassay includes an antibody-antigen reaction and a following turbidity measurement. The third methodology is boronate affinity chromatography, which utilizes a gel matrix containing immobilized boronic acid to capture the cis-diol group of glycated hemoglobin. The variety of Hb Alc testing methodologies requires a novel control that could be used in various methods and devices for detecting Hb Alc levels. In most of the available methods, the first step for measuring Hb Alc levels is the manual or automatic production of a hemolysate by lysing the red blood cells with a special lytic reagent. Therefore, there is an ongoing need for cellular Hb Alc standards or controls that exhibit a similar matrix to that of patient specimens and that function in the analytical testing phases during an Hb Alc assay.
Currently, there are a number of Hb Alc normal and abnormal controls on the market. Some of these controls are disclosed in Wu et. al. U.S. Pat. No. 7,247,484 B2; Wu U.S. Pat. No. 6,890,756 B2; Posner et. al. US Patent Publication 2005/0175977; and Ryan et al. U.S. Pat. No. 7,361,513 B2; all incorporated herein by reference. The Alc-Cellular control preparation described in U.S. Pat. No. 7,361,513 begins by selection of red blood cells from a suitable subject. The Level 1 (lower or normal level) is described to be manufactured by utilizing the red blood cells obtained from a healthy donor with an Alc≦6%. The Level 2 (higher or diabetic level) was described to be manufactured by utilizing the red blood cells obtained from known diabetic donor with an Alc≧9%. The suitable red blood cells were then stabilized and preserved for long term stability. However, manufacturing diabetic level Alc control using the diabetic blood samples, obtained from diabetic donors suffer a number of serious draw backs. These drawbacks include both inferior quality and insufficient quantity of diabetic blood samples, as well as economical disadvantages. Obtaining natural diabetic blood from diabetic patients is restricted. Therefore, obtaining sufficient amount of natural diabetic blood to meet the growing manufacturing volume of Alc control has become an increasing challenge. The availability of diabetic blood sample with a definite range of Alc value is even more difficult. Red Cross eligibility for blood donation states that diabetic individuals can donate blood only if the individual is under treatment and the situations are under control. The Alc values of blood samples obtained from diabetic patients are also inconsistent. According to the American Diabetic Association, any individual with Alc≧6.5 is identified as diabetic. The Alc values of blood samples from diabetic patients may vary from 6.5 to as high as ˜30.0. Further, the lot-to-lot variability of the Alc values of the Alc controls manufactured by mixing these blood samples are extremely high. In other words, different lots of the diabetic level of Alc control may have significantly different Alc values. This inconsistency is much less in the case of normal level due to abundance of blood sample with Alc≈5-6% range. The Alc values of the blood sample obtained from diabetic patient may appear falsely elevated or decreased if the blood of the individual donor contains any abnormal hemoglobin variant. A number of clinical studies reported that the presence of abnormal hemoglobin variants influences Alc values of healthy and diabetic patients, see Bry L, Chen P C, Sacks D B. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin Chem. 2001; 47: 153-163. In general, ion exchange chromatographic and gel electrophoresis methods are affected more than the immunoassay or affinity based methods. In case of ion exchange chromatographic method, when the abnormal variant co-elutes with Hb-Alc, then an increase in Alc value is observed. If the abnormal variant co-elutes with A0 (normal hemoglobin), then an apparent decrease in Alc value is observed. Therefore, manufacturing Alc control using blood from a donor with unknown hemoglobin composition may cause serious risk in the accuracy of the Alc values. Rey et al. reported presence of Hb Seville[α2β281(EF5)Leu→Phe] causes falsely low Alc value when measured on ion-exchange chromatography, see Rey T H del, Conde-Sanchez M, Ropero-Gradilla P et al. Hemoglobin Seville [α2β281(EF5)Leu→Phe] a silent phenotypic variant that interferes in hemoglobin Alc measurement by ion-exchange HPLC method. Clin Biochem. 2011; 44: 933-935. Bergman et al. demonstrated that presence of Hb Stockholm [β7(A4)Glu→Asp] causes falsely low Alc value on Variant II™ chromatography system, see Bergman A C, Beshara S, Byman I, Karim R, Landin B. A new β-chain variant: Hb Stockholm [(β7(A4)Glu→Asp] causes falsely low HbAlc. Hemoglobin. 2009; 33: 137-142. Friess et al. reported that the presence of a novel hemoglobin variant [β66(E10)Lys→Asn] causes a falsely low Alc value measured on cation exchange Tosoh 2.2, see Friess U, Beck A, Kohne E. et al. Novel hemoglobin variant [β66(E10)Lys→Asn], with decreased oxygen affinity, causes falsely low hemoglobin Alc values by HPLC. Clin Chem. 2003; 49: 1412-1415. Chen et. al reported that the Hb-Raleigh [β1Val→Ala] causes false increase in Alc value on ion-exchange, see Chen D, Crimmins D L, Hsu F F, et al. Hemoglobin Raleigh as the cause of a falsely increased hemoglobin Alc in an automated ion-exchange HPLC method. Clin Chem. 1998; 44: 1296-1301. Zhu et al. demonstrated that the presence of HbS in S-β+-thalassemia causes a false Alc values on Bio-Rad Variant II Turbo, see Zhu Y, Williams L M. Falsely elevated hemoglobin Alc due to S-β+-thalassemia interference in Bio-Rad Variant II Turbo HbAlc assay. Clin Chim Acta. 2009; 409: 18-20. Frers et al. observed falsely increased Alc values by HPLC based Tosoh 2.2 for a blood sample that contained Hb Okayama [β2(NA2)His→Gln], see Frers C R, Dorn S, Schmidt W. et al. Falsely increased HbAlc values by HPLC and falsely decreased values by immunoassay lead to identification of Hb Okayama and help in the management of a diabetic patient. Clin Lab. 2000; 46: 569-573. Common hemoglobin variants such as Hb S, Hb J, Hb F or Hb E are also reported by Chu et al. to influence the Alc measurement by Tosoh G7 analyzer, Chu C H, Lam H C, Lee J K. et al. Common hemoglobin variants in southern Taiwan and their effect on the determination of HbAlc by ion-exchange high-performance liquid chromatography. J Clin Med Assoc 2009; 72: 362-367. Immunoassay method based Alc measurements are also known to be affected by the presence of abnormal hemoglobin variants when immune recognition sites of normal Alc or normal hemoglobin are modified by mutation, see Bry L et al, supra. Blood samples collected from the individuals diagnosed with different diseases are also reported to cause inaccurate Alc measurements. Consequently, Alc control manufactured by using blood samples collected from such patients can introduce a great deal of inconsistency in Alc values of the control product. Bannon et al.11 and Engbaek et al.12 reported falsely elevated Alc values measured by ion-exchange chromatography for the patients with uremia, see Bannon P, Lessard F, Lepage R. Glycated hemoglobin in uremic patients as measured by affinity and ion-exchange chromatography. Clin Chem. 1984; 30: 485-486 and Engbaek F, Christensen S E, Jespersen B. Enzyme immunoassay of hemoglobin Alc: Analytical characteristics and clinical performance for patients with diabetes mellitus, with and without uremia. Clin Chem. 1989; 35: 93-97. For such patients, carbamylated derivative of hemoglobin (hemoglobin+urea reaction product) co-elutes with hemoglobin Alc resulting an apparent increase in Alc value in ion-exchange chromatographic methods. Suzuki et al. reported an extremely high Alc value (21%) in a patient and reasoned the false increase in Alc is due to the acute lymphoblastic leukemia, see Suzuki Y, Shichishima T, Yamashiro Y. et al. A patient with acute lymphoblastic leukaemia presenting an extremely high level (21.0%) of HbAlc. Annals Clin Biochem. 2011; 48: 474-477. Danzig et al. reported that the type 1 diabetic patients with glucose-6-phosphatase dehydrogenase deficiency showed falsely decreased Alc values, see Danzig J A, Moser J T, Belfield P. et al. Glucose-6-phosphate dehydrogenase deficiency diagnosed in an adolescent with type 1 diabetes mellitus and hemoglobin Alc discordant with blood glucose measurement. J. Pediatrics 2011: 849-851. Several chemical agents used as drug may bind with hemoglobin variants which can affect Alc measurement. These hemoglobin-drug derivatives can co-elute with Alc in ion-exchange chromatography causing false result. Likewise, they can interfere with antibody recognition in immunoassay method or chemical recognition in affinity method yielding false results. Evidently, blood samples, obtained from the donors who are under treatment, are not suitable for manufacturing Alc control with a consistent Alc value. Aspirin has been identified by a number of researchers to cause false elevation of Alc values resulted from HPLC based analyzers. Aspirin (acetylsalicylic acid) binds with hemoglobin producing acetylated hemoglobin which co-elutes with Alc in HPLC chromatography resulting falsely elevated Alc, see Nathan D M, Francis T B, Palmer J L. Effect of Aspirin on determination of glycosylated hemoglobin. Clin Chem. 1983; 29: 466-469; Bridges K R, Schmidt G J, Jensen M. et al. The acetylation of hemoglobin by aspirin. In vitro and in vivo. J Clin Invest. 1975; 56:201-207; Camargo J L, Stifft J, Gross J L. The effect of aspirin and Vitamins C and E on HbAlc assays. Clin Chim Acta 2006; 372: 206-209; and Weykamp C W, Penders T J, Siebelder C W M, et al. Interference of carbamylated and acetylated hemoglobin in assays f glycohemoglobin by HPLC, electrophoresis, affinity chromatography and enzyme immunoassay. Clin Chem. 1993; 39: 138-142. Gross et al. identified ribavirin and peginterferon alfe-2b therapy for hepatitis C viral infection cause false lowering of Alc values, see Gross B N, Cross B, Foard J C, Falsely low hemoglobin Alc levels in a patient receiving ribavirin and peginerferon alfa-2b for hepatitis C. Pharmacotherapy, 2009; 29: 121-123. Brown et al. reported false low Alc level for the diabetic patients with chronic kidney disease who were undergoing erythropoietin therapy with epoetin alfa and darbepoetin alfa drugs, Brown J N, Kemp D W, Brice K R. Class effect of erythropoietin therapy on hemoglobin Alc in a patient with diabetes mellitus and chronic kidney disease not undergoing hemodialysis, Pharmacotherapy, 2009; 29:468-472. Vitamins C and E are also suggested to yield falsely decreased Alc values, see Suadek C D, Derr R L, Kalyani R R. Assessing glycemia in diabetes using self-monitoring blood glucose and hemoglobin Alc. Clin Rev. 2006; 295:1688-1697 and Schrot R J, Patel K T, Foulis P. Evaluation of inaccuracies in the measurement of glycemia in the laboratory, by glucose meters, and through measurement of hemoglobin Alc. Clin Diabetes 2007; 25: 43-49. Blood samples interact inconsistently with various analytical methods due to presence of different hemoglobin variants, chemical derivatives of hemoglobin or presence of drugs in the patient blood. A majority of these affect ion-exchange based chromatographic methods such as HPLC or electrophoresis. However, immunoassay and affinity based methods are also known to be affected, see Bry L, supra. Inconsistency of the blood samples for Alc control manufacturing can also be introduced by difference of the ages of the samples obtained. Due to the scarcity, diabetic blood samples might be collected as they become available. Therefore, the ages of the blood cells in a collection of blood packs might be different introducing inferior stability and inconsistent integrity of the cells. As the diabetic donors are rare, the prices of the diabetic blood samples are significantly greater than the normal blood samples.
The factors discussed above justify avoiding manufacture high level Alc control using natural diabetic blood. The factors also encourage to obtain healthy blood samples and biosynthetically convert it to a diabetic resembling blood sample with high Alc values. Such a conversion to synthetically increase hemoglobin Alc concentration by in vitro glycation of hemoglobin are known in literature, see Posner A H, Reichenbach D L, Hemoglobin isolation and preparation of glycosylated hemoglobin. US 2005/0175977; Bunn H F, Haney D N, Kamin S. et al. The biosynthesis of human hemoglobin Alc. J Clin Invest. 1976; 57: 1652-1659 and Spicer K M, Allen R C, Hallett D, Buse M G. Synthesis of hemoglobin Alc and related minor hemoglobins by erythrocytes. In vitro study of regulation. J. Clin Invest. 1979; 64: 40-48. However, these conversions were carried out either by pure hemoglobin or by erythrocyte hemolysate. Glycation of hemoglobin within intact red cells are unprecedented. Ryan et al. described two methods of in vitro glycations of hemoglobin with red cells which are less than ideal for manufacturing purpose.1 The first method proposed by Ryan et al. described a reductive glycation of red blood
cells with 3% hemoglobin and 0.5% NaCNBH3. This method yields 2-hydroxylized glycated hemoglobin which is not recognized as Alc by ion-exchange chromatographic or immunoassay methods. Ryan et al. also described slow synthesis of Alc by incubating red blood cells with ˜3% glucose at 4-6° C. which takes ˜50 days to achieve desired high concentration of Alc. It is desirable for efficient manufacturing to develop processes that can be performed faster.
Many prior art solutions require glycosylation outside of the blood cells which limit the usefulness of glycosylated materials in controls. Many Alc controls also fail to produce controls that resemble a true patient blood sample. Such controls cannot be utilized to create universal controls, or controls that test multiple parameters of a patient blood sample. The leakage of hemoglobin from the red blood cells may render the control ineffective for its intended use, thus requiring that the red blood cell membranes be preserved to prevent this undesirable leakage. This can render controls prepared form such red blood cells to not be stable for the desired time frame.
Unlike control products disclosed in the prior art, the present teachings allow for glycosylation to occur within the red blood cells so that the synthesis of Alc also occurs within the red blood cells. Unexpectedly, the present teachings include methods whereby the red blood cell membranes are preserved so that such synthesis occurs without damage to the cell membrane. As a further benefit of glycosylation within the red blood cells, the resulting control contains a more uniform population of glycosylated cells. Many Alc controls also fail to produce controls that resemble a true patient blood sample. Such controls cannot be utilized to create universal controls, or controls that test multiple parameters of a patient blood sample. The present teachings are directed to controls capable of testing multiple elements of a blood sample, which may include various white blood cell populations, nucleated red blood cells, reticulocytes or other blood cell types. In addition, the present teachings provide for one control product that produces consistent data for a number of hematology analyzers, as opposed to requiring a distinct control for each analyzer. As a further benefit of the present teachings, the preservation of the red blood cell membranes results in a shelf-stable product whereby the red blood cells resist hemoglobin leakage for a period of at least about 4 months, or longer. The leakage of hemoglobin from the red blood cells may render the control ineffective for its intended use, thus requiring that the red blood cell membranes be preserved to prevent this undesirable leakage. The present invention addresses one or more of the above needs by providing improved controls and methods for making the controls for testing the Hb Alc level in diabetic blood wherein the controls can be prepared in a reasonable time frame with good accuracy and precision and consistency among the standard methods.